Antibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several ADCs are demonstrating promising clinical efficacy, however due to the complexity of human cancer, tumors become refractory to most drug treatments. We hypothesized that cultured tumor cells chronically treated with an ADC would acquire mechanisms of resistance unique to ADC-based therapy. Human breast cancer cell lines were exposed to multiple cycles of an anti-Her2 trastuzumab-maytansinoid conjugate (TM) at IC80 concentrations for 3 days followed by ∼1 week without treatment to simulate a maximally tolerated dose followed by recovery. After ∼2 months, significant resistance developed in JIMT1 and MDA-MB-361 cell lines. The potency of TM conjugate on drug-selected cell lines was reduced to the activity observed on Her2-negative cells (>20 & >270X in JIMT1 & 361 cell models, respectively). Flow cytometry revealed 58% and 25% decreases in Her2 receptor number in JIMT-TM & 361-TM, respectively. Proteomic profiling of surface proteins in JIMT-TM cells demonstrated significant increases in proteins involved in post-translational modification (e.g., ubiquitinating enzymes, kinases, and phosphatases), as well as elevated levels of endosomal and vesicle proteins (e.g., RAB family members), and proteins mediating microtubule and actin dynamics. Notably, ABC drug transporters were not altered in JIMT-TM cells. In 361-TM cells, an increase in ABCC1 (MRP1) was observed, but no changes in ABCB1 (MDR1) which typically effluxes tubulin inhibitors. These data suggest the acquisition of complex resistance mechanisms upon ADC treatment. The cross-resistance profile of these ADC refractory models was evaluated. Minimal or no resistance (1 - 6X) was observed to free drugs, including maytansine or other standard-of-care tubulin or DNA targeted therapeutics. In JIMT-TM cells, cross-resistance was observed to other trastuzumab-based ADCs, including those containing either non-cleavable or cleavable linkers, and delivering payloads with various biological mechanisms of action. In contrast, 361-TM cells (which were made resistant to an ADC delivering a tubulin inhibitor via a non-cleavable linker) retained significant sensitivity to ADCs containing cleavable linkers, even those containing other tubulin inhibitor-based payloads. Moreover, both 361-TM and JIMT-TM resistant cell lines retained sensitivity to ADCs delivering payloads with alternative (non-tubulin) mechanisms of action. Therefore, despite the reduction in antigen levels observed in both cell lines, modification of the linker and/or the payload was able to overcome resistance mediated by the initial ADC therapy. These data offer the potential to treat refractory tumors with ADCs containing the same antibody vehicle, but delivering alternative linkers or payloads.

Citation Format: Xingzhi Tan, Guixian Jin, Jeremy Myers, Veronica Diesl, Max Follettie, My-Hanh Lam, Sylvia Musto, Kiran Khandke, Manoj Charati, Edmund Graziani, Andreas Maderna, Chakrapani Subramanyam, Frank Koehn, Russell Dushin, Kim Arndt, Christopher J. O'Donnell, Hans-Peter Gerber, Frank Loganzo. Tumor cells selected for resistance to an antibody-drug conjugate retain sensitivity to ADCs with modified linkers and payloads. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4629. doi:10.1158/1538-7445.AM2013-4629